Many tumor types will often initially respond to chemotherapy treatment. However, successful treatment of these diseases is limited by the failure to eliminate minimal residual disease (MRD). Moreover, relapse is often associated with a multi-drug resistant phenotype that contributes to decreased chemotherapy sensitivity and failure of salvage treatment. Acquired drug resistance entails a great deal of genomic complexity, which dramatically increases the difficulty in managing such diseases. The present inventors propose that a viable strategy for enhancing the efficacy of currently used cytotoxics is to identify targets which contribute to de novo drug resistance thereby increasing the efficacy and success of initial therapy intervention and decreasing relapse rates. By definition, de novo drug resistance is represented by mechanisms, which do not require drug selection for the expression of the phenotype. De novo drug-resistant models are in contrast to historical drug-resistant models, which have been based upon unicellular drug selections over time, thereby excluding the impact of tumor cell interaction with the microenvironment on drug response (see FIG. 1).
The present inventors demonstrated that components of the tumor microenvironment influence the response of hematopoietic cell lines to chemotherapeutics and could contribute to de novo resistance. Specifically, it has been shown that adhesion of leukemia and multiple myeloma cell lines to the extracellular matrix component, fibronectin (FN) via β1 integrin, is sufficient to inhibit drug-induced apoptosis. (Hazlehurst L A, et al. Oncogene. 2000; 19:4319-4327; Hazlehurst L A, et al. Cancer Res. 2003; 63:7900-7906; Hazlehurst L A, et al. Blood. 2001; 98:1897-1903; Hazlehurst L A, et al. Cancer Res. 2006; 66:2338-2345; Hazlehurst L A, et al. Cancer Metastasis Rev. 2001; 20:43-50; Hazlehurst L A, et al. Cancer Res. 1999; 59:1021-1028; Hazlehurst L A, et al. Biochem Pharmacol. 1995; 50:1087-1094; Hazlehurst L A, et al. Oncogene. 2003; 22:7396-7402). More recently, it was shown that the cell adhesion induced drug resistance (CAM-DR) phenotype is operative in clinical samples taken from primary multiple myeloma (Hazlehurst L A, et al. Cancer Res. 2003; 63:7900-7906). Together, these data suggest that adhesion of hematopoietic tumor cells to FN could contribute to the failure of currently used cytotoxics to eradicate the entire disease and may facilitate the subsequent emergence of clinical drug resistance.
The emergence of drug-resistant tumor cells remains an obstacle to the successful treatment of many hematopoietic malignancies, including Acute Myeloid Leukemia (AML), Multiple Myeloma, and Chronic Myelogenous Leukemia (CML). Our laboratory, along with others, has previously shown that cell adhesion via β1 integrins is sufficient to cause drug resistance (Chrenek M A, et al. Breast Cancer Res. 2001; 3:224-229; Damiano J S, et al. Blood. 1999; 93:1658-1667; Damiano J S, et al. Leukemia. 2001; 15:1232-1239; de la Fuente M T, et al. J Leukoc Biol. 2002; 71:495-502; Hazlehurst L A, et al. Oncogene. 2000; 19:4319-4327; Hazlehurst L A, et al. Cancer Res. 2003; 63:7900-7906; Hazlehurst L A, et al. Blood. 2001; 98:1897-1903; Sethi T, et al. Nat Med. 1999; 5:662-668; Sherman-Baust C A, et al. Cancer Cell. 2003; 3:377-386).
β1 integrin mediated cell adhesion is a clinically relevant mechanism whereby tumor cells can evade cell death induced by chemotherapy. In support of this hypothesis, the inventors have previously shown that cell adhesion via β1 integrins to the extracellular matrix fibronectin confers a multi-drug resistance phenotype. As indicated above, the inventors have referred to that phenotype as CAM-DR. Moreover, the inventors have validated the CAM-DR phenotype in primary myeloma patient specimens. More recently, the inventors have shown that co-culturing tumor cells with a bone marrow stroma cell whereby tumor cells are in direct contact with the bone marrow stroma cell line confers drug resistance. The present inventors have referred to this phenotype as environmental mediated drug resistance or EMDR. Due to the in vitro findings, the inventors hypothesized that targeting β1 integrin mediated cell adhesion will increase the efficacy of drugs used to treat cancers.
The development of target based drug discovery has provided novel approaches for the treatment of hematopoietic malignancies. However, despite the recent advances in drug discovery it is clear that rationally designed chemotherapeutics such as the BCR-ABL kinase inhibitor imanitib, although effective, do not circumvent the emergence of clinical drug resistance (Buchdunger E, et al. Cancer Res. 1996; 56:100-104; Druker B J, et al. Nat Med. 1996; 2:561-566; Gorre M E, et al. Science. 2001; 293:876-880; Hochhaus A, et al. Science. 2001; 293:2163; Sawyers C L. Science. 2001; 294:1834). These data support the importance of identifying and targeting drug resistant mechanism(s) as a viable strategy for improving the efficacy of cancer chemotherapy. A cancer cell can employ multiple strategies that ultimately favor survival following cytotoxic insult. These mechanisms include decreased drug uptake, increased drug efflux, alterations in the drug target, drug metabolism, repair of DNA damage, cell cycle checkpoint mediators, and changes in downstream mediators of the apoptotic pathway.
Traditionally, drug resistance mechanisms have been identified and functionally characterized in unicellular models. However, unicellular models lack consideration of host-tumor cell interactions that may participate in the emergence of the drug resistant phenotype. We propose that the initial selection pressure for tumor growth and survival is represented by host-tumor cell interactions and furthermore, these same interactions may participate in modulating drug response and emergence of drug resistance. Indeed, Teicher et al provided experimental data supporting this concept (Teicher B A, et al. Science. 1990; 247:1457-1461). These investigators showed that treatment of mice bearing EMT-6 mammary tumors over a six-month period with alkylating agents resulted in the selection of a drug resistance phenotype that was operative only in-vivo. Conversely, the resistance phenotype was not detected in a unicellular tissue culture system indicating the importance of the microenvironment in mediating the expression of a functional drug resistance phenotype. Thus we suggest that in order to identify clinically relevant de-novo drug resistant targets new models must be developed allowing for reconstitution of complex host-tumor cell interactions.
The first model demonstrating the survival effects of cell adhesion was developed by Durand and Sutherland in 1972 (Durand R E, S et al. Exp Cell Res. 1972, 71:75-80). In this model, they demonstrated that forcing V79 Chinese hamster cells to grow in a spheroid culture was causative for radiation resistance relative to cells treated as a monolayer. The implication of the spheroid model is that cell-cell contact may enhance survival of tumor cells in response to cytotoxic stimuli. Unlike solid tumors, most hematopoietic tumors do not grow as a spheroid in-vivo. However, as the knowledge of adhesion receptors, ligands and counter-receptors has grown, it is apparent that hematopoietic cells maintain a dynamic relationship with the bone marrow stroma and components of the extracellular matrix. Furthermore, it is well documented that adhesion molecules participate in the growth, differentiation, survival and homing of hematopoietic cells (Bohnsack J F, et al. Blood. 1994; 83:543-552; Hamdan H F, et al. Oncol Res. 1992; 4:201-207; Potocnik A J, et al. Immunity. 2000; 12:653-663; Wang M W, et al. Cell Growth Differ. 1998; 9:105-112).
Cellular adhesion taken in context within the entire microenvironment is multifactorial. However, of these adhesion molecules identified in hematopoietic cells, integrins are the best characterized for their role in regulating cell growth, survival, differentiation and homing to the bone marrow. Integrin receptors are comprised of non-covalently associated heterodimeric subunits. The integrin receptors contain both an alpha and beta subunit. To date, 17α and 8β subunits have been identified. Among the integrin family, VLA-4 (α4β1) and VLA-5 (α5β1) integrins are the most commonly reported integrin receptors expressed in AML (Vila L, et al. Exp Hematol. 1995; 23:514-518) and multiple myeloma (Cook G, et al. Acta Haematol. 1997; 97:81-89).
The inventors' initial observations using cellular adhesion to FN as a model system for investigating CAM-DR was done in multiple myeloma cell lines (Damiano J S, et al. Blood. 1999; 93:1658-1667). Myeloma is a disease that homes to the bone marrow and is characterized by the accumulation of plasma cells in the bone marrow. But multiple myeloma is not the only hematopoietic malignancy in which CAM-DR may contribute to disease progression and drug resistance. It is well accepted that initial chemotherapy of CML and AML results in rapid clearing of detectable disease in the peripheral blood. However, elimination of disease in the bone marrow is a more arduous task, suggesting that de-novo drug resistance associated with the bone marrow microenvironment may contribute to residual disease. Consistent with these clinical observations, the present inventors shown a similar CAM-DR phenotype in CML and AML cell lines, indicating that adhesion mediated drug resistance may impact several malignancies (Damiano J S, et al. Leukemia. 2001; 15:1232-1239; Hazlehurst L A, et al. Blood. 2001; 98:1897-1903). In addition, Matsunaga et al. recently showed in a mouse model of AML minimal residual disease (MRD) that treatment with a VLA-4 specific antibody and AraC significantly increased survival compared to treatment with AraC alone (Matsunaga T, et al. Nat. Med. 2003; 9:1158-1165). The inventors propose that cell adhesion maybe a key determinant of de-novo drug resistance and failure to eliminate MRD in AML.
Despite recent advances in the treatment of multiple myeloma, the disease remains incurable. Due to the inherent resistance associated with myeloma identification of targets contributing to failure to eliminate minimal residual disease remains in area of active research. As indicated above, myeloma typically homes to the bone marrow, and this specialized environment may contribute to failure to eliminate minimal residual disease. The bone marrow microenvironment represents an area that is enriched in deposition of extracellular matrixes. The inventors previously showed that adhesion of myeloma cells to the extrallular matrix fibronectin (FN) inhibits apoptosis induced by mechanistically and structurally diverse chemotherapeutic agents (Damiano J S et al. Blood, 1999; 93:1658-1667; Hazlehurst L A et al. Oncogene, 2000; 19:4319-4327; Hazlehurst L A et al. Cancer Res., 2003; 63:7900-7906). Moreover, the inventors recently demonstrated that adhesion of primary patient multiple myeloma specimens to FN protects multiple myeloma cells form melphalan induced apoptosis (Hazlehurst L A et al. Cancer Res., 2003; 63:7900-7906). Taken together, our previous data indicates that targeting FN receptors (VLA-4, VLA-5 integrins) and/or signaling may enhance the efficacy of cytotoxic agents used to treat multiple myeloma.
VLA-4 and VLA-5 integrins are the most common integrins expressed on myeloma cells (Van Riet I et al. Br J. Haematol., 1991; 79:421-427). These two integrin receptors are both comprised of a β1 subunit, and experimental evidence indicates that downstream signaling occurs through the β1 integrin cytoplasmic tail region. β1 integrins are reported to activate a complex network of signaling, which includes activation of Mapk, Akt, Fak/Pyk2 and integrin linked kinase or ILK (Astier A et al. J Biol. Chem., 1997; 272:19719-19724; Chen Q et al. J Biol. Chem., 1996; 271:18122-18127; King W G et al. Mol Cell Biol., 1997; 17:4406-4418; Schaller M D and Parsons J T et al. Mol Cell Biol., 1995; 15:2635-2645; Schlaepfer D D et al. Nature, 1994; 372:786-791; Hannigan G E et al. Nature, 1996; 379:91-96). In addition, the present inventors recently showed that adhesion of myeloma and leukemia cells to FN reduced the levels of the pro-apoptotic Bcl-2 family member Bim (Hazlehurst L A et al. British Journal Haematology, 2007; 136(2):269-75).